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Molecular Psychiatry Feb 2021Mutations in AUTS2 are associated with autism, intellectual disability, and microcephaly. AUTS2 is expressed in the brain and interacts with polycomb proteins, yet it is...
Mutations in AUTS2 are associated with autism, intellectual disability, and microcephaly. AUTS2 is expressed in the brain and interacts with polycomb proteins, yet it is still unclear how mutations in AUTS2 lead to neurodevelopmental phenotypes. Here we report that when neuronal differentiation is initiated, there is a shift in expression from a long isoform to a short AUTS2 isoform. Yeast two-hybrid screen identified the splicing factor SF3B1 as an interactor of both isoforms, whereas the polycomb group proteins, PCGF3 and PCGF5, were found to interact exclusively with the long AUTS2 isoform. Reporter assays showed that the first exons of the long AUTS2 isoform function as a transcription repressor, but the part that consist of the short isoform acts as a transcriptional activator, both influenced by the cellular context. The expression levels of PCGF3 influenced the ability of the long AUTS2 isoform to activate or repress transcription. Mouse embryonic stem cells (mESCs) with heterozygote mutations in Auts2 had an increase in cell death during in vitro corticogenesis, which was significantly rescued by overexpressing the human AUTS2 transcripts. mESCs with a truncated AUTS2 protein (missing exons 12-20) showed premature neuronal differentiation, whereas cells overexpressing AUTS2, especially the long transcript, showed increase in expression of pluripotency markers and delayed differentiation. Taken together, our data suggest that the precise expression of AUTS2 isoforms is essential for regulating transcription and the timing of neuronal differentiation.
Topics: Animals; Cell Differentiation; Cytoskeletal Proteins; Exons; Mice; Neurons; Phenotype; Protein Isoforms; Transcription Factors
PubMed: 30953002
DOI: 10.1038/s41380-019-0409-1 -
Cells Apr 2022Vascular endothelial growth factor A (VEGF-A) is a secreted protein that stimulates angiogenesis in response to hypoxia. Under hypoxic conditions, a non-canonical long...
Vascular endothelial growth factor A (VEGF-A) is a secreted protein that stimulates angiogenesis in response to hypoxia. Under hypoxic conditions, a non-canonical long isoform called is concomitantly expressed with . Once translated, L-VEGF is proteolytically cleaved to generate N-VEGF and VEGF-A. Interestingly, while VEGF-A is secreted and affects the surrounding cells, N-VEGF is mobilized to the nucleus. This suggests that N-VEGF participates in transcriptional response to hypoxia. In this study, we performed a series of complementary experiments to examine the functional role of N-VEGF. Strikingly, we found that the mere expression of N-VEGF followed by its hypoxia-independent mobilization to the nucleus was sufficient to induce key genes associated with angiogenesis, such as isoforms, as well as genes associated with cell survival under hypoxia. Complementarily, when was genetically depleted, key hypoxia-induced genes were downregulated and cells were significantly susceptible to hypoxia-mediated apoptosis. This is the first report of N-VEGF serving as an autoregulatory arm of VEGF-A. Further experiments will be needed to determine the role of N-VEGF in cancer and embryogenesis.
Topics: Apoptosis; Cell Hypoxia; Humans; Hypoxia; Neovascularization, Pathologic; Protein Isoforms; Vascular Endothelial Growth Factor A
PubMed: 35455969
DOI: 10.3390/cells11081289 -
Philosophical Transactions of the Royal... Nov 2023Protein isoforms, generated through alternative splicing or promoter usage, contribute to tissue function. Here, we characterize the expression of predicted and...
Protein isoforms, generated through alternative splicing or promoter usage, contribute to tissue function. Here, we characterize the expression of predicted and isoforms in hair follicles and describe expression of , a hitherto unknown PADI2 isoform, in the oligodendrocyte lineage. transcription is initiated from a downstream intronic promoter, generating an N-terminally truncated, unstable, PADI2β. By contrast to the established role of the canonical PADI2 (PADI2α) (Falcao . 2019 . , 1090-1102.e10. (doi:10.1016/j.celrep.2019.03.108)), PADI2β inhibits oligodendrocyte differentiation, suggesting that PADI2 isoforms exert opposing effects on oligodendrocyte lineage progression. We localize and to developing hair follicles and find that both transcripts are expressed at low levels in progenitor cells, only to increase in expression concomitant with differentiation. When expressed , PADI3α and PADI3β are enriched in the cytoplasm and precipitate together. Whereas PADI3β protein stability is low and PADI3β fails to induce protein citrullination, we find that the enzymatic activity and protein stability of PADI3α is reduced in the presence of PADI3β. We propose that PADI3β modulates PADI3α activity by direct binding and heterodimer formation. Here, we establish expression and function of and isoforms, expanding on the mechanisms in place to regulate citrullination in complex tissues. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.
Topics: Protein-Arginine Deiminases; Cell Differentiation; Protein Isoforms
PubMed: 37778375
DOI: 10.1098/rstb.2022.0451 -
Bioinformatics (Oxford, England) Mar 2020Isoforms are alternatively spliced mRNAs of genes. They can be translated into different functional proteoforms, and thus greatly increase the functional diversity of...
MOTIVATION
Isoforms are alternatively spliced mRNAs of genes. They can be translated into different functional proteoforms, and thus greatly increase the functional diversity of protein variants (or proteoforms). Differentiating the functions of isoforms (or proteoforms) helps understanding the underlying pathology of various complex diseases at a deeper granularity. Since existing functional genomic databases uniformly record the annotations at the gene-level, and rarely record the annotations at the isoform-level, differentiating isoform functions is more challenging than the traditional gene-level function prediction.
RESULTS
Several approaches have been proposed to differentiate the functions of isoforms. They generally follow the multi-instance learning paradigm by viewing each gene as a bag and the spliced isoforms as its instances, and push functions of bags onto instances. These approaches implicitly assume the collected annotations of genes are complete and only integrate multiple RNA-seq datasets. As such, they have compromised performance. We propose a data integrative solution (called DisoFun) to Differentiate isoform Functions with collaborative matrix factorization. DisoFun assumes the functional annotations of genes are aggregated from those of key isoforms. It collaboratively factorizes the isoform data matrix and gene-term data matrix (storing Gene Ontology annotations of genes) into low-rank matrices to simultaneously explore the latent key isoforms, and achieve function prediction by aggregating predictions to their originating genes. In addition, it leverages the PPI network and Gene Ontology structure to further coordinate the matrix factorization. Extensive experimental results show that DisoFun improves the area under the receiver operating characteristic curve and area under the precision-recall curve of existing solutions by at least 7.7 and 28.9%, respectively. We further investigate DisoFun on four exemplar genes (LMNA, ADAM15, BCL2L1 and CFLAR) with known functions at the isoform-level, and observed that DisoFun can differentiate functions of their isoforms with 90.5% accuracy.
AVAILABILITY AND IMPLEMENTATION
The code of DisoFun is available at mlda.swu.edu.cn/codes.php?name=DisoFun.
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Computational Biology; Gene Ontology; Molecular Sequence Annotation; Protein Isoforms; ROC Curve
PubMed: 32176770
DOI: 10.1093/bioinformatics/btz847 -
BMC Genomics 2015Protein-protein interactions (PPIs) are key to understanding diverse cellular processes and disease mechanisms. However, current PPI databases only provide...
BACKGROUND
Protein-protein interactions (PPIs) are key to understanding diverse cellular processes and disease mechanisms. However, current PPI databases only provide low-resolution knowledge of PPIs, in the sense that "proteins" of currently known PPIs generally refer to "genes." It is known that alternative splicing often impacts PPI by either directly affecting protein interacting domains, or by indirectly impacting other domains, which, in turn, impacts the PPI binding. Thus, proteins translated from different isoforms of the same gene can have different interaction partners.
RESULTS
Due to the limitations of current experimental capacities, little data is available for PPIs at the resolution of isoforms, although such high-resolution data is crucial to map pathways and to understand protein functions. In fact, alternative splicing can often change the internal structure of a pathway by rearranging specific PPIs. To fill the gap, we systematically predicted genome-wide isoform-isoform interactions (IIIs) using RNA-seq datasets, domain-domain interaction and PPIs. Furthermore, we constructed an III database (IIIDB) that is a resource for studying PPIs at isoform resolution. To discover functional modules in the III network, we performed III network clustering, and then obtained 1025 isoform modules. To evaluate the module functionality, we performed the GO/pathway enrichment analysis for each isoform module.
CONCLUSIONS
The IIIDB provides predictions of human protein-protein interactions at the high resolution of transcript isoforms that can facilitate detailed understanding of protein functions and biological pathways. The web interface allows users to search for IIIs or III network modules. The IIIDB is freely available at http://syslab.nchu.edu.tw/IIIDB.
Topics: Algorithms; Alternative Splicing; Cluster Analysis; Computational Biology; Databases, Protein; Genome, Human; Humans; Internet; Protein Interaction Mapping; Protein Interaction Maps; Protein Isoforms; Proteins; Reproducibility of Results; Software
PubMed: 25707505
DOI: 10.1186/1471-2164-16-S2-S10 -
Cell Death & Disease Mar 2024The advancement of RNAseq and isoform-specific expression platforms has led to the understanding that isoform changes can alter molecular signaling to promote... (Review)
Review
The advancement of RNAseq and isoform-specific expression platforms has led to the understanding that isoform changes can alter molecular signaling to promote tumorigenesis. An active area in cancer research is uncovering the roles of ubiquitination on spliceosome assembly contributing to transcript diversity and expression of alternative isoforms. However, the effects of isoform changes on functionality of ubiquitination machineries (E1, E2, E3, E4, and deubiquitinating (DUB) enzymes) influencing onco- and tumor suppressor protein stabilities is currently understudied. Characterizing these changes could be instrumental in improving cancer outcomes via the identification of novel biomarkers and targetable signaling pathways. In this review, we focus on highlighting reported examples of direct, protein-coded isoform variation of ubiquitination enzymes influencing cancer development and progression in gastrointestinal (GI) malignancies. We have used a semi-automated system for identifying relevant literature and applied established systems for isoform categorization and functional classification to help structure literature findings. The results are a comprehensive snapshot of known isoform changes that are significant to GI cancers, and a framework for readers to use to address isoform variation in their own research. One of the key findings is the potential influence that isoforms of the ubiquitination machinery have on oncoprotein stability.
Topics: Humans; Ubiquitination; Protein Isoforms; Gastrointestinal Neoplasms; Carcinogenesis; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases
PubMed: 38453895
DOI: 10.1038/s41419-024-06575-z -
Nature Communications Sep 2023Shotgun proteomics is essential for protein identification and quantification in biomedical research, but protein isoform characterization is challenging due to the...
Shotgun proteomics is essential for protein identification and quantification in biomedical research, but protein isoform characterization is challenging due to the extensive number of peptides shared across proteins, hindering our understanding of protein isoform regulation and their roles in normal and disease biology. We systematically assess the challenge and opportunities of shotgun proteomics-based protein isoform characterization using in silico and experimental data, and then present SEPepQuant, a graph theory-based approach to maximize isoform characterization. Using published data from one induced pluripotent stem cell study and two human hepatocellular carcinoma studies, we demonstrate the ability of SEPepQuant in addressing the key limitations of existing methods, providing more comprehensive isoform-level characterization, identifying hundreds of isoform-level regulation events, and facilitating streamlined cross-study comparisons. Our analysis provides solid evidence to support a widespread role of protein isoform regulation in normal and disease processes, and SEPepQuant has broad applications to biological and translational research.
Topics: Humans; Proteomics; Protein Isoforms; Biomedical Research; Carcinoma, Hepatocellular; Liver Neoplasms
PubMed: 37726316
DOI: 10.1038/s41467-023-41558-2 -
Nature Protocols Feb 2021Although mammalian embryo development depends on critical protein isoforms that arise from embryo-specific nucleic acid modifications, the role of these isoforms is not...
Although mammalian embryo development depends on critical protein isoforms that arise from embryo-specific nucleic acid modifications, the role of these isoforms is not yet clear. Challenges arise in measuring protein isoforms and nucleic acids from the same single embryos and blastomeres. Here we present a multimodal technique for performing same-embryo nucleic acid and protein isoform profiling (single-embryo nucleic acid and protein profiling immunoblot, or snapBlot). The method integrates protein isoform measurement by fractionation polyacrylamide gel electrophoresis (fPAGE) with off-chip analysis of nucleic acids from the nuclei. Once embryos are harvested and cultured to the desired stage, they are sampled into the snapBlot device and subjected to fPAGE. After fPAGE, 'gel pallets' containing nuclei are excised from the snapBlot device for off-chip nucleic acid analyses. fPAGE and nuclei analyses are indexed to each starting sample, yielding same-embryo multimodal measurements. The entire protocol, including processing of samples and data analysis, takes 2-3 d. snapBlot is designed to help reveal the mechanisms by which embryo-specific nucleic acid modifications to both genomic DNA and messenger RNA orchestrate the growth and development of mammalian embryos.
Topics: Animals; Blastocyst; Blastomeres; DNA; Electrophoresis, Polyacrylamide Gel; Embryo, Mammalian; Embryonic Development; Female; Immunoblotting; Mice; Nucleic Acids; Protein Isoforms; RNA, Messenger
PubMed: 33452502
DOI: 10.1038/s41596-020-00449-2 -
Molecular Cancer Research : MCR Apr 2015The small GTPase Ras is mutated in about 20% of human cancers, primarily at active site amino acid residues G12, G13, and Q61. Thus, structural biology research has... (Review)
Review
The small GTPase Ras is mutated in about 20% of human cancers, primarily at active site amino acid residues G12, G13, and Q61. Thus, structural biology research has focused on the active site, impairment of GTP hydrolysis by oncogenic mutants, and characterization of protein-protein interactions in the effector lobe half of the protein. The C-terminal hypervariable region has increasingly gained attention due to its importance in H-Ras, N-Ras, and K-Ras differences in membrane association. A high-resolution molecular view of the Ras-membrane interaction involving the allosteric lobe of the catalytic domain has lagged behind, although evidence suggests that it contributes to isoform specificity. The allosteric lobe has recently gained interest for harboring potential sites for more selective targeting of this elusive "undruggable" protein. The present review reveals critical insight that isoform-specific differences appear prominently at these potentially targetable sites and integrates these differences with knowledge of Ras plasma membrane localization, with the intent to better understand the structure-function relationships needed to design isoform-specific Ras inhibitors.
Topics: Catalytic Domain; Cell Membrane; Crystallography, X-Ray; Humans; Models, Molecular; Protein Binding; Protein Isoforms; Protein Structure, Secondary; ras Proteins
PubMed: 25566993
DOI: 10.1158/1541-7786.MCR-14-0535 -
Current Topics in Microbiology and... 2022Macrophages display an array of activation phenotypes depending on the activation signal and the cellular microenvironment. The type and magnitude of the response depend...
Macrophages display an array of activation phenotypes depending on the activation signal and the cellular microenvironment. The type and magnitude of the response depend on signaling molecules as well as on the epigenetic and metabolic status of the cells at the time of activation. The AKT family of kinases consists of three isoforms encoded by independent genes possessing similar functions and structures. Generation of research tools such as isoform-specific gene deletion mutant mice and cells and isoform-specific antibodies has allowed us to understand the role of each kinase isoform in macrophage activation and homeostasis. This chapter discusses the current evidence on the role of AKT kinases in macrophage activation, polarization, and homeostasis, highlighting the gaps in knowledge and future challenges in the field.
Topics: Animals; Macrophage Activation; Macrophages; Mice; Protein Isoforms; Proto-Oncogene Proteins c-akt; Signal Transduction
PubMed: 36243844
DOI: 10.1007/978-3-031-06566-8_7